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1.
J Contemp Dent Pract ; 23(4): 383-387, 2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-35945829

RESUMO

AIM: The aim of the study was to compare the ability of three endodontic sealers, Endofill (END), AH Plus (AHP), and Sealer Plus BC (SPB), to penetrate dentinal tubules. MATERIALS AND METHODS: Forty-five human teeth, single-rooted and previously instrumented mandibular premolars, were randomly divided into three experimental groups (n = 15): END (n = 15), AHP (n = 15), and SPB (n = 15). After obturation, dental sections were performed horizontally, at 2 and 5 mm from the root apex. The samples were analyzed by scanning electron microscopy associated with cathodoluminescence. Percentage penetration (PP%) and maximum penetration depth (MPD) of the sealers were evaluated by the Kruskal-Wallis and Mann-Whitney tests, for general and paired data, respectively. The Wilcoxon test was applied to analyze the differences between the 5 and 2 mm distances. A 5% significance level was adopted. RESULTS: As for PP%, AHP and SPB were similar (p = 0.127) and presented higher values than END (AHP, p = 0.024 and SPB, p <0.001); with regard to MPD, AHP and SPB did not differ either (p = 0.450), but were higher than END (p <0.001); in both analyses, penetration was greater at 5 mm than at 2 mm (p <0.001). CONCLUSION: SPB showed satisfactory performance in penetrating dentinal tubules, being similar to AHP, and superior to END. CLINICAL SIGNIFICANCE: Greater penetration of sealer into the dentinal tubules may increase the chance of successful endodontic treatment.


Assuntos
Materiais Restauradores do Canal Radicular , Dente Pré-Molar , Resinas Epóxi , Humanos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Obturação do Canal Radicular
3.
Neurosurg Focus ; 52(6): E9, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35921184

RESUMO

OBJECTIVE: Communication between neurosurgeons and pathologists is mandatory for intraoperative decision-making and optimization of resection, especially for invasive masses. Handheld confocal laser endomicroscopy (CLE) technology provides in vivo intraoperative visualization of tissue histoarchitecture at cellular resolution. The authors evaluated the feasibility of using an innovative surgical telepathology software platform (TSP) to establish real-time, on-the-fly remote communication between the neurosurgeon using CLE and the pathologist. METHODS: CLE and a TSP were integrated into the surgical workflow for 11 patients with brain masses (6 patients with gliomas, 3 with other primary tumors, 1 with metastasis, and 1 with reactive brain tissue). Neurosurgeons used CLE to generate video-flow images of the operative field that were displayed on monitors in the operating room. The pathologist simultaneously viewed video-flow CLE imaging using a digital tablet and communicated with the surgeon while physically located outside the operating room (1 pathologist was in another state, 4 were at home, and 6 were elsewhere in the hospital). Interpretations of the still CLE images and video-flow CLE imaging were compared with the findings on the corresponding frozen and permanent H&E histology sections. RESULTS: Overall, 24 optical biopsies were acquired with mean ± SD 2 ± 1 optical biopsies per case. The mean duration of CLE system use was 1 ± 0.3 minutes/case and 0.25 ± 0.23 seconds/optical biopsy. The first image with identifiable histopathological features was acquired within 6 ± 0.1 seconds. Frozen sections were processed within 23 ± 2.8 minutes, which was significantly longer than CLE usage (p < 0.001). Video-flow CLE was used to correctly interpret tissue histoarchitecture in 96% of optical biopsies, which was substantially higher than the accuracy of using still CLE images (63%) (p = 0.005). CONCLUSIONS: When CLE is employed in tandem with a TSP, neurosurgeons and pathologists can view and interpret CLE images remotely and in real time without the need to biopsy tissue. A TSP allowed neurosurgeons to receive real-time feedback on the optically interrogated tissue microstructure, thereby improving cross-functional communication and intraoperative decision-making and resulting in significant workflow advantages over the use of frozen section analysis.


Assuntos
Glioma , Telepatologia , Endoscopia/métodos , Humanos , Lasers , Microscopia Confocal/métodos
4.
Klin Lab Diagn ; 67(7): 407-413, 2022 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-35924771

RESUMO

The development of mycotic colonization of the base surface with further biodegradation of acrylic plastics is currently of undoubted interest. The oral cavity is a favorable ecological niche for colonization by fungi and their subsequent possible invasion into the epithelium of the oral mucosa. The method of modulation interference laser microscopy is of considerable interest to researchers in medicine in the context of obtaining the necessary information about the morphological characteristics of microbial cells and the microbiome community as a whole during the colonization of a certain ecological niche in the human body. Purpose of the study: to analyze the microrelief of the biofilm of yeast-like fungi of the species Candida albicans of base plastics of the hot type of polymerization using the method of laser modulation interference microscopy. An experimental study was carried out in order to study biofilms of yeast-like fungi of the genus Candida on samples of basic plastics, an image of a biofilm of yeast-like fungi of the species Candida albicans was obtained on the surface of a plastic of a hot type of polymerization (polymethyl methacrylate) in the visualization of the phase portrait, a description of its horizontal and vertical bioprofile. As a result of the research, the heterogeneous structure of the biofilm was determined, due to the different density and accumulation of cells along the surface, the characteristics of the surface were established in accordance with the roughness criteria. The microrelief parameters on a separately arbitrarily selected section line allow one to determine the characteristics of the biofilm in the required area and make it possible to judge the nature of its formation in a certain biological niche.


Assuntos
Candida albicans , Plásticos , Biofilmes , Humanos , Lasers , Microscopia Confocal , Microscopia de Interferência
5.
Cutis ; 109(5): 269-271, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35856764

RESUMO

The diagnosis of a small-diameter melanoma may be challenging. We report the case of a 57-year-old man with a small pigmented papular lesion (2.5-mm diameter) that was suspicious on dermoscopy. A more confident differential diagnosis between an atypical nevus and a melanoma was necessary for correct management. Reflectance confocal microscopy (RCM) allowed a confident diagnosis in this lesion, which was an invasive melanoma with 0.3-mm Breslow thickness. This case highlights the benefit of RCM to reach a confident diagnosis and correct management of a small-diameter invasive melanoma.


Assuntos
Melanoma , Neoplasias Cutâneas , Dermoscopia , Diagnóstico Diferencial , Humanos , Masculino , Melanoma/diagnóstico , Melanoma/patologia , Microscopia Confocal , Pessoa de Meia-Idade , Neoplasias Cutâneas/diagnóstico , Neoplasias Cutâneas/patologia
6.
Methods Mol Biol ; 2473: 15-22, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35819755

RESUMO

We hereby describe a method to image cargo trafficking from the cis- to the trans-face of the Golgi apparatus. Briefly, we combine nocodazole treatment that breaks down the Golgi ribbon, temperature blocks that slow down cargo transport, and a drug-controlled aggregation system that controls the size of the cargo and its retention at different stages of the secretory pathway. Using this method, we first position the cargo within the cis-face of the Golgi. When traffic resumes upon temperature block release, kinetics of transport can be assessed by confocal microscopy through colocalization of the cargo with cis- and trans-Golgi markers. This method allows for testing various modes of intra-Golgi transports and can be adapted to investigate other steps of the secretory pathway.


Assuntos
Complexo de Golgi , Via Secretória , Complexo de Golgi/metabolismo , Cinética , Microscopia Confocal
7.
Biomater Sci ; 10(16): 4525-4537, 2022 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-35788579

RESUMO

Doxorubicin is an anthracycline drug most commonly used in cancer therapy. It intercalates with the nuclear DNA and induces toxicity by causing DNA breaks and histone eviction. However, the kinetics of its action on the nucleus has not been mapped effectively. This study shows successful PEGylation and DOX loading through π-π interaction onto carbogenic fluorescent nanodots (FNDs), which have an affinity for the nucleolus. Then the drug release from the nanoparticle and its action on the nuclear environment were aptly mapped using both fluorescence lifetime imaging and superresolution radial fluctuation (SRRF) techniques. Here for the first time, the nuclear degradation kinetics caused by the released DOX from the FNDs as a result of DNA double-strand breaks and histone eviction was visualized. This led to the observation of decreasing length, breadth, and complex structure of the nuclear clusters from 6 h to 24 h, resulting in isolated cluster visualization. However, the superresolution images for free DOX and untreated cells reveal no such drastic effects at the same concentration and time points, unlike DOX loaded particles.


Assuntos
Doxorrubicina , Histonas , DNA , Fragmentação do DNA , Doxorrubicina/química , Microscopia Confocal , Polietilenoglicóis/química
8.
Org Biomol Chem ; 20(29): 5812-5819, 2022 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-35838007

RESUMO

The synthesis of the fluorescent organic carbon monoxide releasing molecules oCOm-57, oCOm-58, and oCOm-66 are reported. These oCOms are water soluble and exhibit a "turn-on" fluorescent behaviour when CO is released under physiological conditions. oCOm-66 also contains an additional nitro-naphthalimide moiety that functions as a fluorescent reporter. Delivery of CO released from these oCOms to the mitochondria of AC-16 cardiomyocytes was confirmed using confocal microscopy in conjuction with MitoTracker Red. While the neutral, PEGylated oCOm-57 was found to remain in the extracellular environment releasing CO to diffuse into the cellular compartments, the positively charged oCOm-58 and -66 are targeted to the mitochondria where they release CO. Notably, the use of the fluorescent oCOms in live cellular imaging, allows the intracellular CO delivery and oCOm localisation to be characterised. This cellular confocal study also shows that, subtoxic concentrations of CO released from these molecules preserved mitochondrial energetics as indicated by the membrane potential dependent MitoTracker Red.


Assuntos
Monóxido de Carbono , Mitocôndrias , Corantes Fluorescentes/farmacologia , Microscopia Confocal , Naftalimidas/farmacologia
9.
Ocul Surf ; 25: 155-162, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35872076

RESUMO

PURPOSE: To study changes in the subbasal nerve plexus by In vivo confocal microscopy (IVCM) in Sjögren's Syndrome (SS) with or without associated Small Fiber Neuropathy (SFN), in order to prevent diagnostic delay. METHODS: Seventy-one patients with SS, including 19 with associated SFN, 20 healthy volunteers and 20 patients with Meibomian gland dysfunction (MGD) were included in this retrospective case-control study. IVCM was used to investigate subbasal nerve plexus density and morphology. RESULTS: Corneal sensitivity as evaluated with the Cochet-Bonnet aesthesiometer was significantly reduced in the SS group versus the control group (P = 0.026) and the MGD group (P = 0.037). The number of inflammatory cells was significantly increased in the SS group to 86.2 ± 82.1 cells/mm2 compared to the control group (P < 0.001). The density of the subbasal nerve plexus was significantly reduced to 16.7 ± 6.5 mm/mm2 in the SS group compared to the control group (P < 0.005) and the MGD group (P = 0.042). The tortuosity of the nerves in the SS group was significantly increased compared to the control group (P < 0.001) and the MGD group (P = 0.025). The average number of subbasal nerve plexus neuromas was significantly increased in the SS group compared to the control group (P = 0.001), with a significant increase in the average number of neuromas in SS patients with associated SFN compared to SS patients without SFN (P = 0.008). CONCLUSION: IVCM can be useful to detect corneal nerve changes in SS patients and may allow earlier diagnosis of the disease and to consider new therapeutic approaches.


Assuntos
Neuroma , Síndrome de Sjogren , Neuropatia de Pequenas Fibras , Estudos de Casos e Controles , Córnea/inervação , Diagnóstico Tardio , Humanos , Microscopia Confocal , Neuroma/complicações , Nervo Oftálmico , Estudos Retrospectivos , Síndrome de Sjogren/complicações , Síndrome de Sjogren/diagnóstico , Neuropatia de Pequenas Fibras/complicações
10.
J Biomed Opt ; 27(7)2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35879817

RESUMO

SIGNIFICANCE: Reflectance confocal microscopy (RCM) is a noninvasive, in vivo technology that offers near histopathological resolution at the cellular level. It is useful in the study of phenomena for which obtaining a biopsy is impractical or would cause unnecessary tissue damage and trauma to the patient. AIM: This review covers the use of RCM in the study of skin and the use of machine learning to automate information extraction. It has two goals: (1) an overview of information provided by RCM on skin structure and how it changes over time in response to stimuli and in disease and (2) an overview of machine learning approaches developed to automate the extraction of key morphological features from RCM images. APPROACH: A PubMed search was conducted with additional literature obtained from references lists. RESULTS: The application of RCM as an in vivo tool in dermatological research and the biologically relevant information derived from it are presented. Algorithms for image classification to epidermal layers, delineation of the dermal-epidermal junction, classification of skin lesions, and demarcation of individual cells within an image, all important factors in the makeup of the skin barrier, were reviewed. Application of image analysis methods in RCM is hindered by low image quality due to noise and/or poor contrast. Use of supervised machine learning is limited by time-consuming manual labeling of RCM images. CONCLUSIONS: RCM has great potential in the study of skin structures. The use of artificial intelligence could enable an easier, more reproducible, precise, and rigorous study of RCM images for the understanding of skin structures, skin barrier, and skin inflammation and lesions. Although several attempts have been made, further work is still needed to provide a definite gold standard and overcome issues related to image quality, limited labeled datasets, and lack of phenotype variability in available databases.


Assuntos
Neoplasias Cutâneas , Inteligência Artificial , Epiderme/patologia , Humanos , Microscopia Confocal/métodos , Pele/diagnóstico por imagem , Pele/patologia , Neoplasias Cutâneas/patologia
11.
Nano Lett ; 22(15): 6454-6461, 2022 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-35792810

RESUMO

A recent addition to the toolbox of super-resolution microscopy methods is fluorescence-lifetime single-molecule localization microscopy (FL-SMLM). The synergy of SMLM and fluorescence-lifetime imaging microscopy (FLIM) combines superior image resolution with lifetime information and can be realized using two complementary experimental approaches: confocal-laser scanning microscopy (CLSM) or wide-field microscopy. Here, we systematically and comprehensively compare these two novel FL-SMLM approaches in different spectral regions. For wide-field FL-SMLM, we use a commercial lifetime camera, and for CLSM-based FL-SMLM we employ a home-built system equipped with a rapid scan unit and a single-photon detector. We characterize the performances of the two systems in localizing single emitters in 3D by combining FL-SMLM with metal-induced energy transfer (MIET) for localization along the third dimension and in the lifetime-based multiplexed bioimaging using DNA-PAINT. Finally, we discuss advantages and disadvantages of wide-field and confocal FL-SMLM and provide practical advice on rational FL-SMLM experiment design.


Assuntos
DNA , Imagem Individual de Molécula , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Nanotecnologia , Imagem Individual de Molécula/métodos
12.
Phys Rev Lett ; 128(24): 248002, 2022 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-35776445

RESUMO

We show that mixing a colloidal gel with larger, non-Brownian grains generates novel flow-switched bistability. Using a combination of confocal microscopy and rheology, we find that prolonged moderate shear results in liquefaction by collapsing the gel into disjoint globules, whereas fast shear gives rise to a yield-stress gel with granular inclusions upon flow cessation. We map out the state diagram of this new "mechanorheological material" with varying granular content and demonstrate that its behavior is also found in separate mixture using different particles and solvents.


Assuntos
Reologia , Microscopia Confocal
13.
J Biomed Opt ; 27(6)2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35773774

RESUMO

SIGNIFICANCE: Raman spectroscopy (RS) provides an automated approach for assisting Mohs micrographic surgery for skin cancer diagnosis; however, the specificity of RS is limited by the high spectral similarity between tumors and normal tissues structures. Reflectance confocal microscopy (RCM) provides morphological and cytological details by which many features of epidermis and hair follicles can be readily identified. Combining RS with deep-learning-aided RCM has the potential to improve the diagnostic accuracy of RS in an automated fashion, without requiring additional input from the clinician. AIM: The aim of this study is to improve the specificity of RS for detecting basal cell carcinoma (BCC) using an artificial neural network trained on RCM images to identify false positive normal skin structures (hair follicles and epidermis). APPROACH: Our approach was to build a two-step classification model. In the first step, a Raman biophysical model that was used in prior work classified BCC tumors from normal tissue structures with high sensitivity. In the second step, 191 RCM images were collected from the same site as the Raman data and served as inputs for two ResNet50 networks. The networks selected the hair structure and epidermis images, respectively, within all images corresponding to the positive predictions of the Raman biophysical model with high specificity. The specificity of the BCC biophysical model was improved by moving the Raman spectra corresponding to these selected images from false positive to true negative. RESULTS: Deep-learning trained on RCM images removed 52% of false positive predictions from the Raman biophysical model result while maintaining a sensitivity of 100%. The specificity was improved from 84.2% using Raman spectra alone to 92.4% by integrating Raman spectra with RCM images. CONCLUSIONS: Combining RS with deep-learning-aided RCM imaging is a promising tool for guiding tumor resection surgery.


Assuntos
Carcinoma Basocelular , Aprendizado Profundo , Neoplasias Cutâneas , Carcinoma Basocelular/diagnóstico por imagem , Dermoscopia/métodos , Humanos , Microscopia Confocal/métodos , Neoplasias Cutâneas/patologia
14.
Sci Rep ; 12(1): 11167, 2022 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-35778532

RESUMO

We aimed to investigate the density and morphology of corneal dendritic cells (DCs) in dry eye (DE) patients with or without Sjogren's syndrome (SS). This study included 28 patients with Sjogren's syndrome dry eye (SSDE), 33 patients with non-Sjogren's syndrome dry eye (NSSDE), and 30 age and sex matched healthy volunteers. In vivo confocal microscopy (IVCM) was used to investigate density and morphology (size, dendrites, and field) of DC. Compared with NSSDE and healthy group, SSDE showed significantly higher DC density, larger DC size, more DC dendrites with larger DC field (all P < 0.001). Comparison between NSSDE and healthy group demonstrated that DC density, dendrites and field were significantly higher in NSSDE. However, there was no significant difference in DC size (P = 0.076). DC density and morphological parameters showed significant associations with the systemic severity (salivary gland biopsy and serum antibodies) and ocular surface damage. The corneal epithelium DC density and morphological alterations were obvious in SSDE, which reflected higher level of immune activation and inflammatory response in SS. Marked correlations were found between DC density/morphology and systemic/ocular severity. Dynamic assessment of corneal DC may facilitate to clarify pathogenesis, stratify patient, and tailor treatment in SS patients.


Assuntos
Síndromes do Olho Seco , Epitélio Corneano , Síndrome de Sjogren , Contagem de Células , Células Dendríticas/metabolismo , Síndromes do Olho Seco/patologia , Epitélio Corneano/metabolismo , Humanos , Microscopia Confocal , Síndrome de Sjogren/patologia
15.
Sensors (Basel) ; 22(13)2022 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-35808232

RESUMO

The phase separation and aggregation of proteins are hallmarks of many neurodegenerative diseases. These processes can be studied in living cells using fluorescent protein constructs and quantitative live-cell imaging techniques, such as fluorescence recovery after photobleaching (FRAP) or the related fluorescence loss in photobleaching (FLIP). While the acquisition of FLIP images is straightforward on most commercial confocal microscope systems, the analysis and computational modeling of such data is challenging. Here, a novel model-free method is presented, which resolves complex spatiotemporal fluorescence-loss kinetics based on dynamic-mode decomposition (DMD) of FLIP live-cell image sequences. It is shown that the DMD of synthetic and experimental FLIP image series (DMD-FLIP) allows for the unequivocal discrimination of subcellular compartments, such as nuclei, cytoplasm, and protein condensates based on their differing transport and therefore fluorescence loss kinetics. By decomposing fluorescence-loss kinetics into distinct dynamic modes, DMD-FLIP will enable researchers to study protein dynamics at each time scale individually. Furthermore, it is shown that DMD-FLIP is very efficient in denoising confocal time series data. Thus, DMD-FLIP is an easy-to-use method for the model-free detection of barriers to protein diffusion, of phase-separated protein assemblies, and of insoluble protein aggregates. It should, therefore, find wide application in the analysis of protein transport and aggregation, in particular in relation to neurodegenerative diseases and the formation of protein condensates in living cells.


Assuntos
Doenças Neurodegenerativas , Proteínas , Recuperação de Fluorescência Após Fotodegradação/métodos , Humanos , Microscopia Confocal , Microscopia de Fluorescência/métodos , Fotodegradação , Transporte Proteico
16.
Technol Cancer Res Treat ; 21: 15330338221093149, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35790459

RESUMO

Purpose: To assess the safety and technical feasibility of in-vivo needle-based forward-looking confocal laser endomicroscopy in prostate tissue. Methods: For this feasibility study, 2 patients with a suspicion of prostate cancer underwent transperineal needle-based confocal laser endomicroscopy during ultrasound-guided transperineal template mapping biopsies. After intravenous administration of fluorescein, needle-based confocal laser endomicroscopy imaging was performed with a forward-looking probe (outer diameter 0.9 mm) in 2 trajectories during a manual push-forward and pullback motion. A biopsy was taken in a coregistered parallel adjacent trajectory to the confocal laser endomicroscopy trajectory for histopathologic comparison. Peri- and postprocedural adverse events, confocal laser endomicroscopy device malfunction and procedural failures were recorded. Needle-based confocal laser endomicroscopy image quality assessment, image interpretation, and histology were performed by an experienced confocal laser endomicroscopy rater and uro-pathologist, blinded to any additional information. Results: In both patients, no peri- and post-procedural adverse events were reported following needle-based confocal laser endomicroscopy. No confocal laser endomicroscopy device malfunction nor procedural failures were reported. Within 1.5 min after intravenous administration of fluorescein, needle-based confocal laser endomicroscopy image quality was sufficient for interpretation for at least 14 min, yielding more than 5000 confocal laser endomicroscopy frames per patient. The pullback confocal laser endomicroscopy recordings and most of the push-forward recordings almost only visualized erythrocytes, being classified as non-representative. During the push-forward recordings, prostate tissue was occasionally visualized in single frames, insufficient for histopathologic comparison. Prostate carcinoma was identified by biopsy in one patient (Gleason score 4 + 3 = 7, >50%), while the biopsy from the other patient showed no malignancy. Conclusion: Needle-based confocal laser endomicroscopy imaging of in-vivo prostate tissue with a forward-looking confocal laser endomicroscopy probe is safe without device malfunctions or procedural failures. Needle-based confocal laser endomicroscopy is technically feasible, but the acquired confocal laser endomicroscopy datasets are non-representative. The confocal laser endomicroscopy images' non-representative nature is possibly caused by bleeding artifacts, movement artifacts and a lack of contact time with the tissue of interest. A different confocal laser endomicroscopy probe or procedure might yield representative images of prostatic tissue.


Assuntos
Próstata , Neoplasias da Próstata , Estudos de Viabilidade , Fluoresceínas , Humanos , Biópsia Guiada por Imagem , Lasers , Masculino , Microscopia Confocal/métodos , Próstata/diagnóstico por imagem , Neoplasias da Próstata/diagnóstico por imagem
17.
Sci Rep ; 12(1): 12277, 2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35853990

RESUMO

Acquiring detailed 3D images of samples is needed for conducting thorough investigations in a wide range of applications. Doing so using nondestructive methods such as X-ray computed tomography (X-ray CT) has resolution limitations. Destructive methods, which work based on consecutive delayering and imaging of the sample, face a tradeoff between throughput and resolution. Using focused ion beam (FIB) for delayering, although high precision, is low throughput. On the other hand, mechanical methods that can offer fast delayering, are low precision and may put the sample integrity at risk. Herein, we propose to use femtosecond laser ablation as a delayering method in combination with optical and confocal microscopy as the imaging technique for performing rapid 3D imaging. The use of confocal microscopy provides several advantages. First, it eliminates the 3D image distortion resulting from non-flat layers, caused by the difference in laser ablation rate of different materials. It further allows layer height variations to be maintained within a small range. Finally, it enables material characterization based on the processing of material ablation rate at different locations. The proposed method is applied on a printed circuit board (PCB), and the results are validated and compared with the X-ray CT image of the PCB part.


Assuntos
Imageamento Tridimensional , Terapia a Laser , Imageamento Tridimensional/métodos , Lasers , Microscopia Confocal/métodos , Tomografia Computadorizada por Raios X/métodos
18.
Phys Med Biol ; 67(14)2022 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-35732165

RESUMO

Objective.Corneal nerve fiber (CNF) has been found to exhibit morphological changes associated with various diseases, which can therefore be utilized to aid in the early diagnosis of those diseases. CNF is usually visualized under corneal confocal microscopy (CCM) in clinic. To obtain the diagnostic biomarkers from CNF image produced from CCM, image processing and quantitative analysis are needed. Usually, CNF is segmented first and then CNF's centerline is extracted, allowing for measuring geometrical and topological biomarkers of CNF, such as density, tortuosity, and length. Consequently, the accuracy of the segmentation and centerline extraction can make a big impact on the biomarker measurement. Thus, this study is aimed to improve the accuracy and universality of centerline extraction.Approach.We developed a new thinning algorithm based on neighborhood statistics, called neighborhood-statistics thinning (NST), to extract the centerline of CNF. Compared with traditional thinning and skeletonization techniques, NST exhibits a better capability to preserve the fine structure of CNF which can effectively benefit the biomarkers measurement above. Moreover, NST incorporates a fitting process, which can make centerline extraction be less influenced by image segmentation.Main results.This new method is evaluated on three datasets which are segmented with five different deep learning networks. The results show that NST is superior to thinning and skeletonization on all the CNF-segmented datasets with a precision rate above 0.82. Last, NST is attempted to be applied for the diagnosis of keratitis with the quantitative biomarkers measured from the extracted centerlines. Longer length and higher density but lower tortuosity were found on the CNF of keratitis patients as compared to healthy patients.Significance.This demonstrates that NST has a good potential to aid in the diagnostics of eye diseases in clinic.


Assuntos
Algoritmos , Fibras Nervosas , Córnea/diagnóstico por imagem , Humanos , Processamento de Imagem Assistida por Computador/métodos , Microscopia Confocal
19.
Brain Struct Funct ; 227(6): 1933-1947, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35643821

RESUMO

The mirror technique adapted for electron microscopy allows correlating neuronal structures across the cutting plane of adjoining light microscopic sections which, however, have a limited thickness, typically less than 100 µm (Talapka et al. in Front Neuroanat, 2021, https://doi.org/10.3389/fnana.2021.652422 ). Here, we extend the mirror technique for tissue blocks in the millimeter range and demonstrate compatibility with serial block-face electron microscopy (SBEM). An essential step of the methodological improvement regards the recognition that unbound resin must be removed from the tissue surface to gain visibility of surface structures. To this, the tissue block was placed on absorbent paper during the curing process. In this way, neuronal cell bodies could be unequivocally identified using epi-illumination and confocal microscopy. Thus, the layout of cell bodies which were cut by the sectioning plane can be correlated with the layout of their complementary part in the adjoining section processed for immunohistochemistry. The modified mirror technique obviates the spatial limit in investigating synaptology of neurochemically identified structures such as neuronal processes, dendrites and axons.


Assuntos
Imageamento Tridimensional , Neurônios , Axônios , Imageamento Tridimensional/métodos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Neurônios/ultraestrutura
20.
STAR Protoc ; 3(3): 101483, 2022 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-35769923

RESUMO

Quantitative 3D imaging of organ-wide cellular and subcellular components is central for revealing and understanding complex interactions between stem cells and their microenvironment. Here, we present a gentle but fast whole-mount immunofluorescence staining protocol for 3D confocal microscopy (iFAST3D) that preserves the 3D structure of the entire tissue and that of subcellular structures with high fidelity. The iFAST3D protocol enables reproducible and high-resolution 3D imaging of stem cells and various niche components for many mouse organs and tissues. For complete details on the use and execution of this protocol, please refer to Saçma et al. (2019).


Assuntos
Imageamento Tridimensional , Células-Tronco , Animais , Imageamento Tridimensional/métodos , Camundongos , Microscopia Confocal/métodos , Coloração e Rotulagem
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